Autism spectrum disorder – NDA collection 2962

NDA collection 2962 utilized de-identified postmortem human brain tissues that were collected by the University of Maryland Brain and Tissue Bank (an NIH NeuroBioBank; institutional review board (IRB) no. HP-00042077), the Autism BrainNet (McLean IRB nos. 2006-P-0001161 and 2006-P-0001862; Western IRB no. 20141029), and the LIBD (Maryland Dept of Health and Mental Hygiene IRB no. 12–14; Western IRB no. 20111080) following provision of informed consent. The secondary use of these tissues for genomic studies was approved by the Boston Children’s Hospital Institutional Review Board (protocol S07-02-0087).

The majority of the data of collection 2962 is composed of high depth whole genome sequencing (≥210X coverage) of either the DLPFC or the prefrontal cortex tissue of 59 ASD-affected and 15 unaffected subjects. Additional tissues that underwent WGS include two occipital lobe samples of controls and the saliva of parents of two affected individuals. DNA extraction was performed with the QIAamp DNA Mini kit lysis buffer, phenol chloroform extraction, and isopropanol workup.

One of two methods was applied to obtain high depth WGS data. The first method involved sequencing of seven libraries per sample produced by the Illumina TruSeq DNA PCR-Free library prep kit on an Illumina HiSeqX Ten platform to yield a combined coverage of ≥210X. The second method used sequencing of a single library produced with the Illumina TruSeq Nano DNA library kit on an Illumina HiSeqX Ten for 200X coverage in one run (Supp. Fig. 1)⁵.

The data were aligned to GRCh37d5 using bwa mem 0.7.8^15–17. Variant calling was performed with Mutect-Panel-of-Normals according to GATK 3.5 best practices^18,20. Point mutation variants identified within segmental duplications and non-diploid regions were filtered from the results. The Genome Aggregation Database was used to set a threshold for filtering variants at a maximum population minor allele frequency above 1 × 10^−5 21 Population polymorphisms were also removed²². Variants outside a variant allele frequency between 0.02 to 0.40 were also filtered. MosaicForecast was used for phasing and identifying postzygotic variants⁹.